Substituted benzhydrylamines as handles for solid phase peptide synthesis

ABSTRACT

The present invention is directed to a novel &#34;handle&#34; for solid phase peptide synthesis. The handles can be converted from a stable form to a labile form, allowing for cleavage of a peptide amide from the support after synthesis under mild conditions, and after deprotection of the amino acid side chains. The handles are based on a substituted benzhydrylamine skeleton.

This is a continuation of application Ser. No. 08/137,048, filed Jul. 5,1994, now U.S. Pat. No. 5,684,131 which is the national stage ofInternational Application no. PCT/US92/02962, filed Apr. 10, 1992.

1. FIELD OF THE INVENTION

The present invention is directed to a novel "handle" for solid phasepeptide synthesis. The handles can be converted from a stable form to alabile form, allowing for cleavage of a peptide from the support aftersynthesis under mild conditions, and after deprotection of theprotecting groups from the amino acid side chains. The handles are basedon a substituted benzhydrylamine skeleton.

2. BACKGROUND OF THE INVENTION

As used in the art of peptide synthesis, the handle is defined as abifunctional spacer that serves to link the peptide fragment or firstamino acid to the polymer support. Many solid phase resins and handlesare available in the art, as is described in Fields and Noble, 1990,Int. J. Pept. Protein Res. 35:161-214. To be useful, handles must bestable to the reaction conditions of peptide synthesis, but when thesynthesis is complete, the handle needs to allow for cleavage of thepeptide from the solid support.

A few handles that can be converted from a stable form of handle to alabile form have been designed and synthesized. Unfortunately, thesehandles have found no broad application in practice. For example, the4-benzylthiophenyl- and 4-benzylsulfonyl-handle (D. L. Marshall, I. E.Liener: J. Org. Chem. 35, 867 (1970)) and sulfonamide-handle (G. W.Kenner, J. R. McDermott, R. C. Sheppard: Chem. Commun. 636, (1971)) havenot found broad application. Further examples can be found in "ThePeptides, Analysis, Synthesis, Biology," E. Gross, J. Meienhofer, Eds.,Academic Press New York, vol. 2. p. 88 (1980), vol. 3. p. 209 (1981).Quite recently, the handle based on the 4-alkylthiobenzyl alcohol hasbeen disclosed (EP 274,998 and EP 274,999; Chem. Abstr. 110, 135705(1989)) and used in peptide synthesis. After reduction of the sulfoxidemoiety, the 4-alkylthiobenzyl ester is cleaved to give a peptide withfree carboxyl group.

The majority of aforementioned handles have, however, certain obviouslimitations concerning the amino acid residues that can be affected bythe conversion step. As a rule, oxidation steps cannot be used when thepeptide contains sensitive amino acids such as tryptophan, cysteine,cystine, and methionine. Another problem is possible methylation during"activation" of handle and long cleavage times which can cause thedamage of peptide.

Moreover, the handles presently available are not well adapted to bothBoc and Fmoc synthetic procedures.

3. SUMMARY OF THE INVENTION

The instant invention is directed to substituted benzhydrylamine handlesfor peptide synthesis. The handles are prepared and used in acid labileform or in stable form. The stable form can be converted to an acidlabile form for cleavage of the peptide from the resin.

The substituted benzhydrylamine handles are of the general formula (I):##STR1## in which a=1 to 3, X¹ is S!R¹ or X¹ is Z, the X¹ groups are inthe ortho or para positions of the first benzene ring, and R¹ is analkyl group and

in which b=0 to 3, X² is S!R², the X² groups are in the ortho or parapositions of the second benzene ring, and R² is an alkyl group; and

in which Z is R³, OR³ or S!R³ in any position not occupied by X¹ unlessX¹ is Z, and R³ is an alkyl group comprising a reactive functional groupfor coupling to a solid phase support, and in which if X¹ is Z, Z isS!R³ in an ortho or para position; and

in which c=0 or 1 and d=0 or 1, Y is OR⁴, and R⁴ is an alkyl group; and

in which S! is G, and G is --S--, --SO-- or --SO₂ --; and

in which D is H, a protecting group or an N.sup.α -protected acyl. Asused herein, "alkyl" can be a C₁ to C₁₀.

Further provided are methods for synthesis of the handles of theinvention. In one embodiment, hydroxy or mercapto benzophenones arereacted with ω-haloesters of alkanecarboxylic acids in the presence offluoride ions. The benzophenone carbonyl is subsequently converted to anamine by routine synthetic methods, e.g., reaction with hydroxylamine toyield an oxime, followed by reduction, e.g., with zinc, to yieldbenzhydrylamine, or by reductive amination, e.g., by reaction withammonium formate. Alternatively the benzophenone can be reduced to thealcohol and amidated with an N.sup.α -protected amino acid amide.

The present invention offers distinct advantages over earlier methods ofcreating stable handles for peptide synthesis that can be treated afterthe synthetic reaction is complete to make the handle labile. Once thehandle is converted from stable form to labile form, the peptide can beconveniently cleaved from a solid phase support and used. The peptide iscleaved in a carboxy amide (rather than free carboxyl) form. Theoxidized handles, i.e., in the sulfoxide or sulfone form, areextraordinarily stable in acidic media, e.g., trifluoroacetic acid, aswell as conditions generally used in peptide synthesis. When reduced tosulfide form, the handle is acid labile. In a preferred embodiment, thesulfoxide or sulfone is readily reduced to sulfide, to convert thestable handle to a labile form.

One particular advantage of the handles of the invention is that theyallow for deprotection of the amino acid side chains prior to cleavage.Under standard solid phase synthesis, the cations generated duringdeprotection and cleavage are available to react with the cleavedpeptide. Often reaction of these cations with the peptide results indecreased yield and purity. According to the present invention, thecations generated during deprotection can be washed away since thedeprotected peptide remains on the solid support. During cleavage, thecations remain with the solid support, away from the cleaved peptide.

Another advantage of the invention is that cleavage of the peptide afterreduction of the handle can be performed under much milder conditionsthan are generally available. For example, in a standard Boc synthesiscleavage requires treatment with the powerful acid, usually HF. Thepresent invention thus allows a Boc synthesis with the final cleavageunder much milder conditions.

Yet a further advantage of the invention is that the handle can be usedfor both Fmoc and Boc synthetic strategies.

Yet another advantage of the handles of the invention is that cleavageyields a C-terminal peptide amide under less drastic conditions than arecurrently available.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to handles for peptide synthesis thatare stable, but that can be converted to labile form for subsequentcleavage of the peptide from the solid phase support. The stability ofthe handles is determined by the oxidation state of an alkyl-sulfursubstituent.

The substituted benzhydrylamine handles are of the general formula (I):##STR2## in which a=1 to 3, X¹ is S!R¹ or X¹ is Z, the X¹ groups are inthe ortho or para positions of the first benzene ring, and R¹ is analkyl group; and

in which b=0 to 3, X² is S!R², the X² groups are in the ortho or parapositions of the second benzene ring, and R² is an alkyl group; and

in which Z is R³, OR³ or S!R³ in any position not occupied by X¹ unlessX¹ is Z, and R³ is an alkyl group comprising a reactive functional groupfor coupling to a solid phase support, and in which if X¹ is Z, Z isS!R³ in an ortho or para position; and

in which c=0 or 1 and d=0 or 1, Y is OR⁴, and R⁴ is an alkyl group; and

in which S! is G, and G is --S--, --SO-- or --SO₂ --; and

in which D is H, a protecting group or an N.sup.α -protected acyl. Asused herein, "alkyl" can be a C₁ to C₁₀.

As used herein, the term alkyl includes but is not limited to C₁ toabout C₁₀ alkane, alkene and alkyne (i.e., saturated and unsaturatedhydrocarbons). For example, an alkyl group may be methyl, ethyl,ethenyl, propyl, propenyl, propynyl, etc. The term alkyl as used hereinincludes branched chain as well as linear chain groups, and cyclicalkyl.

As used herein, the term "handle" is defined as a bifunctional spacer.The handle has one functional group that binds to a solid phase support.The handle has an second functional group that can be conjugated to anamino acid or a peptide. The handle provides for cleavage of the peptideafter synthesis is complete.

As used herein, the term "solid phase support" is not limited to aspecific type of support. Rather a large number of supports areavailable and are known to one of ordinary skill in the art. Solid phasesupports include silica gels, resins, derivatized plastic films, glassbeads, cotton, plastic beads, alumina gels. A suitable solid phasesupport may be selected on the basis of desired end use and suitabilityfor various synthetic protocols. For example, for peptide synthesis,solid phase support may refer to resins such as p-methylbenzhydrylamine(pMBHA) resin (Peptides International, Louisville, Ky.), polystyrene(e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories,etc.), poly (dimethylacrylamide)-grafted styrene co-divinylbenzene(e.g., POLYHIPE® resin, obtained from Aminotech, Canada), polyamideresin (obtained from Peninsula Laboratories), polystyrene resin graftedwith polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany)or polydimethylacrylamide resin (obtained from Milligen/Biosearch,California).

In one embodiment, the solid phase support may be suitable for in vivouse, i.e., it may serve as a carrier for or support for directapplications of the peptide (e.g., TentaGel, Rapp Polymere, Tubingen,Germany). In a particular embodiment, the solid phase support may bepalatable and orally consumable.

The term "peptide" is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other the bonds, e.g., ester,ether, etc. As used herein the term "amino acid" refers to eithernatural and/or unnatural or synthetic amino acids, including glycine andboth the D and L optical isomers, and amino acid analogs andpeptidomimetics. As used herein, a peptidomimetic is a molecule thatexhibits properties similar to a peptide without having a peptidechemical structure. The peptides may comprise D-amino acids, acombination of D- and L-amino acids, and various "designer" amino acids(e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl aminoacids, etc.) to convey special properties to peptides. Additionally, byassigning specific amino acids at specific coupling steps, peptides withα-helices, β turns, β sheets, γ-turns, and cyclic peptides can begenerated.

4.1. HANDLES

The benzhydrylamine handles of the invention are substituted with atleast one sulfur-containing derivative selected from the groupconsisting of sulfide (SR¹), sulfoxide (SOR¹), and sulfone (SO₂ R¹), inwhich R¹ is an alkyl group of 1 to about 10 carbon atoms. The sulfide,sulfoxide or sulfone derivative is designated throughout thisapplication as S!R. The sulfur-containing group is found at the orthoposition or para position on at least one benzene ring. Twosulfur-containing groups on a single benzene ring can be found at theortho-para or both ortho positions. Three sulfur containing groups canbe found at the two ortho and one para positions. The benzene rings canbe symmetrically substituted with sulfur-containing groups, but need notbe. Preferably both benzene rings contain a sulfur-containing group atthe para or ortho position.

The handle further comprises a linker, Z, for attachment to a solidphase support, i.e., resin, for solid phase peptide synthesis using thehandle. In one embodiment, the linker can be a sulfur-containing group (S!R³) located in the ortho or para position of one benzene ring. Inanother embodiment, the linker can be an alkyl group (R³), alkoxy (OR³)group, or sulfur containing group ( S!R³) located at the ortho, para ormeta position, with the proviso that if Z contains O or S and is in themeta position, it cannot be considered to influence the stabilityproperties of the handle. The alkyl group on Z, R³, has from 1 to about10 carbon atoms, and comprises a functional group, for example,carboxylic acid, for attachment to the resin.

In a further embodiment, the handle comprises an alkoxy group (OR⁴), Y,in the ortho or para positions on one or both benzene rings not occupiedby a sulfur-containing group X. No group Y is found on a ring that has Zin the ortho or para position when Z is OR³ (alkoxy). The alkoxy groupscan be but need not be symmetrically arranged on the two benzene rings.If an alkoxy group (Y) is present, the number of Y groups on a singlebenzene ring is less than or equal to the number of X groups on thering; the total number of Y is less than or equal to the total number ofX groups on the benzhydryl amine, and is preferably less. The alkylportion of Y has 1 to about 10 carbon atoms.

Although the present invention is not limited to any particularmechanism, it is believed that the properties of the handle aredetermined by the electron donating or electron withdrawingcharacteristics of substituents in the ortho or para positions of thebenzene ring or rings. Thus when electron donating groups, such assulfide or alkoxy groups, are in the para or ortho positions, acidolysisof the N--C bond readily occurs because electrons can be donated tostabilize the resulting benzhydryl cation. Conversely, when an electronwithdrawing group such as sulfoxide or sulfone is present in the para orortho positions, acidolysis cannot occur. Since electron donation occursless readily from the meta position, substituents in the meta positionare expected to have orders of magnitude less effect than substituentsin the para or ortho positions.

Substitution with both sulfoxide or sulfone substituents and alkoxysubstituents results in a balance of electron withdrawing and electrondonating groups. However, when the number of electron withdrawingoxidized sulfur-containing groups is at least equal to and preferablygreater than the number of alkoxy groups, the electron withdrawingnature of the oxidized sulfur-containing groups is expected to affectthe properties of the handle to a greater degree. The alkoxy groups areadvantageous for cleaving the handle after reduction of thesulfur-containing groups because of their electron donating properties.

It is believed that the stability of the oxidized handle and the acidlability of the reduced handle are enhanced by increasing the number ofsulfur-containing substituents on one or both benzene rings. Thus theincreased electron withdrawing nature of a handle with more than oneoxidized sulfur-containing group may result in greater chemicalstability of the handle. Similarly, the increased electron donatingpotential of more than one reduced sulfur-containing groups mayfacilitate cleavage under mild conditions.

Additionally, oxidation of the sulfur-containing group to yield asulfone is expected to result in a more stable handle than oxidation tothe sulfoxide because the sulfone has greater electron withdrawingproperties. Since either the sulfone or the sulfoxide can be reduced toyield a sulfide, either oxidation state is suitable for handles of theinvention. Presently, the sulfoxide oxidation state is preferred.

In a further embodiment, the handle can be prepared with a protectinggroup at the amine nitrogen. Suitable protecting groups includetert-butoxycarbonyl (Boc) and N-9-fluorenylmethyloxy carbonyl (Fmoc), toname a few. Any amine protecting group suitable for peptide synthesis issuitable. Many are described in Greene and Wuts, Protective Groups inOrganic Synthesis, Wiley Interscience: New York, 1991, pp. 309-362; andFields and Noble 1990, Int. J. Pept. Protein Res. 35: 161-214. TheN-protected benzhydrylamine can be coupled to the solid support withoutconcern for reactivity of the amine group. After the handle is coupledto the solid support, the protecting group can be removed by routinechemistry, e.g., treatment with piperidine in dimethylformamide in thecase of Fmoc, or treatment with trifluoracetic acid in the case of Boc.

In another embodiment, the handle can be prepared as a benzhydrylamide,i.e., with an acyl group on the amine. Acyl groups for use in theinvention include but are not limited to N.sup.α -protected amino acids,which can be deprotected for subsequent elongation of a peptide chain.The term amino acids is used here as defined above, and includes D and Lamino acids, glycine, and non-naturally occurring amino acids or aminoacid analogs.

When X (or Z) is found as a sulfoxide or sulfone group on the handle,the handle is extremely resistant to acidic media. Reduction of asulfoxide or sulfone to a sulfide yields a handle that is labile toacidolysis. For example, such a handle can be cleaved in trifluoroaceticacid. Thus, the present invention provides handles that are acid stableor acid labile, and provides for conversion of the acid stable form tothe labile form, and vice versa.

4.2. SYNTHESIS OF SUBSTITUTED BENZYHDRYLAMINES

The substituted benzhydrylamines of the present invention can beprepared according to the following methods.

4.2.1. Preparation of Benzophenones

Substituted benzophenones for use as starting materials for synthesis ofhandles of the invention can be prepared synthetically, for example by acondensation reaction of a substituted benzoic acid with a substitutedbenzene, i.e., acylation of benzene.

Suitable substituted benzoic acids include but are not limited to a2-alkylthiobenzoic acid, 4-alkylthiobenzoic acid,2,4-di(alkylthio)benzoic acid, 2,4,6-tri(alkylthio)benzoic acid, 2-(or4-)alkylthio-4(or 2-)alkoxybenzoic acid, 2,4-(or 2,6-alkylthio)-6(or4)-alkoxybenzoic acid, and the like. Moreover, the benzoic acid may besubstituted at the 2,3 or 4 position with an alkyl group comprising afunctional group for coupling to the solid phase support (i.e., Z).Special care must be taken to protect the functional group present onthe alkyl group to prevent its reaction during formation of thebenzophenone. For example, if the functional group is a carboxylic acid,it must be protected from reaction with thionyl chloride. Alternativelythe benzoic acid can be substituted at the 2, 3 or 4 position with ahydroxy or mercapto (i.e., sulfhydryl) group. The hydroxy or mercaptogroup is preferably at the 2 or 4 position. If the substituted benzoicacid includes a 2 or 4 mercapto substituent, it need not include analkylthio substituent since upon alkylation, the mercapto group willbecome an alkylthio group with the required properties. In a specificebodiment, the substituted benzoic acid is2-hydroxy-4-(methylthio)benzoic acid.

Suitable substituted benzene groups include but are not limited toalkylthiobenzene (e.g., thioanisole), 1,3-dialkylthiobenzene,1,3,5-trialkylthiobenzene, 3-alkoxy-alkylthiobenzene (e.g.,3-alkoxythioanisole), 5-alkoxy-1,3-dialkylthiobenzene and3-alkoxy-1,5-dialkylthiobenzene. In a specific embodiment, thesubstituted benzene is thioanisole.

In a further embodiment, if the benzoic acid lacks an alkyl group foruse as a linker, and lacks a hydroxy or mercapto group suitable foralkylation to attach the linker, the substituted benzene can includeeither of these groups.

Many variations are possible for the substituted benzoic acid andsubstituted benzene that can be used to prepare the benzophenone.Although many combinations and permutations are possible, and arecontemplated by the instant invention, often the simplest startingmaterials are preferred, since these are generally less expensive,easier to obtain, and less susceptible to undesirable side reactions.Thus, the substituted benzoic acid and substituted benzene chosen mustform a benzophenone with at least one sulfur-containing substituent in apara or ortho position on a benzene ring, preferably a sulfur-containingsubstituent on both benzene rings, and a linker or a functional groupsuitable for attachment of a linker.

In a specific embodiment, infra, a substituted benzoic acid is heated inthionyl chloride to yield the corresponding benzoic acid chloride.Generally this reaction can be run by heating the benzoic acid in abouttwo molar equivalents of thionyl chloride to about 40° C. for about 10min to about 30 min with stirring. The benzoyl chloride is thendissolved (at about 0.1 to about 1M) in an aprotic solvent, e.g.,ethylene dichloride, at about 40° C. This solution is mixed with anequimolar amount of a substituted benzene, e.g., thianisole, dissolvedat about 0.2 to about 3M in an aprotic solvent, (e.g., dichloroethane).The mixture is cooled to 0° C., and treated with an equivalent ofaluminum chloride. The reaction mixture is allowed to warm to about 20°C., at which point a second equivalent of AlCl₃ can be added whiletemperature is controlled. The reaction mixture is stirred for about 1to 2 hours at about 40°-50° C., and worked up by standard methods, e.g.,extraction, recrystallization, etc.

It is also contemplated that a benzophenone can be modified by additionof sulfur-containing groups to the ortho or para positions, or bysubstitution of sulfur for oxygen in alkoxy-substituted benzophenones.The exchange of a sulfide for an alkoxy group occurs by treatment of thehydroxy group with chlorodimethylthiocarbamate followed by thermalrearrangement and S-alkylation. This alternative, however, requires useof an asymmetrically substituted benzophenone to provide for attachmentof, or alternatively to act as, a single linker for attachment to thesolid phase support.

4.2.2. Alkylation of Hydroxy or Mercapto-substituted Benzophenones

In the case in which the benzophenone is prepared without an alkyllinking group, alkylation to add the linking group is required. Anyalkylation procedure known in the art can be used in the practice of theinstant invention. These alkylation procedures are accomplished byactivation of an alkyl with a halogen, especially bromine or chlorine,or with another good leaving group. However, where alkylation at theortho position is contemplated, an agent must be added to disrupthydrogen bonding. In a preferred embodiment, the agent is fluoride ion(F⁻).

In a specific embodiment, a substituted benzophenone is dissolved in apolar aprotic solvent. The benzophenone is substituted with a hydroxy ormercapto (SH) group at the 2, 3 or 4 position on one benzene ring.Preferably the concentration of the benzophenone is 5 mM to 1M, morepreferably 50 mM to 0.5M. The optimal concentration depends on thechoice of polar aprotic solvent, and the solubility of the benzophenonetherein. Suitable solvents include but are not limited to acetonitrile,dimethyl formamide, dioxane, and the like, as well as mixtures thereof.

When the hydroxy or mercapto group is in the 2-position, fluoride ionsare present in the solution to facilitate the alkylation reaction. Theions can be provided on an inert support. In a specific embodiment,infra, the fluoride is provided as a suspension of potassium fluoride onalumina. In another embodiment, fluoride is provided astetraethylammonium fluoride.

To this solution is added an alkylating agent, e.g., an ω-haloester ofalkylcarboxylic acid, which can be prepared by halogenation of thecorresponding commercially available alkylcarboxylic acid. In a specificembodiment, methyl bromovalerate is added. The alkylating agent ispreferably added in molar excess relative to the concentration of thebenzophenone, preferably greater than about a 20% excess, morepreferably greater than about a 40% excess. The alkylation reaction canrequire at least about 96 hours for completion at room temperature. Thealkylated benzophenone can be purified by routine methods, e.g.,chromatography or crystallization, or it can be separated from solidmaterials (e.g., if alumina is present) concentrated, e.g., in vacuo,and used without further purification.

4.2.3. Conversion of the Benzophenone to a Benzhydrylamine

The carbonyl group of the substituted benzophenones is converted to anamine group. Conversion of a carbonyl to an amine can be accomplished byany method known in the art, including but not limited to oximeformation followed by reduction, or reductive amination, e.g., withammonium formate. In a specific embodiment, infra, the benzophenone isreacted with hydroxylamine to yield an oxime. This reaction is generallyperformed in a polar solvent, preferably an alcohol, e.g., ethanol,although methanol, propanol, 2-propanol, etc., can also be used with thesubstituted benzophenone at roughly the same concentration as is usedfor the alkylation reaction. Anhydrous weak base, e.g., sodium acetate,can also be present. The reaction mixture is heated to reflux for about1 to about 15 hours, preferably about 7 hours. The mixture can be leftto stand, e.g., overnight, or worked up immediately. In work-up thereaction mixture can be filtered and concentrated in vacuo to yield aproduct that can be used without further purification.

The oxime product is dissolved in alcohol (preferably ethanol) and 25%ammonia at about a 1:3 ratio in about the same concentration as thealkylated benzophenone was used in the oxime step. To this solution isadded about a 10-fold molar excess of powdered zinc. The suspension isheated to 50° C. and stirred for about 48 hours. The suspension isfiltered and concentrated in vacuo to yield the substituted benzhydrylamine.

The benzyhydrylamine obtained by reduction of the oxime is sparinglysoluble or insoluble in polar solvents. Moreover, when the reductionreaction is performed over zinc, zinc remains in the reaction mixture.Thus prior to oxidation of the sulfur-containing substituents(alkylsulfides) and protection of the amino group, it is necessary topurify the benzhydrylamine. Purification procedures include but are notlimited to chromatography, e.g., on silica gel, recrystallization,preparative thin layer chromatography and the like.

In a specific embodiments, a recrystallization procedure is used. Theinsoluble benzhydrylamine is solubilized by adding toluene sulfonicacid, thus forming the acid salt of the amine. This reaction can beperformed by dissolving the benzhydrylamine in an appropriate solvent,e.g., methanol, ethanol, and the like, preferably ethanol, at about100-500 mM, preferably at about 150 mM. A saturated solution oftoluenesulfonic acid (TsOH) in the solvent can be added, preferably by acontrolled addition, e.g., dropwise addition. The product is dissolvedin a polar, protic solvent system that includes water (e.g., an alcohol,such as methanol, and water, 3:1 v/v), and if necessary heated, toincrease solubility. After the solution has cooled to about 20° C., anoxidizing agent, e.g. sodium periodate (to form the sulfoxide) orhydrogen peroxide (to form the sulfone) is added in molar excess basedon equivalents of S atoms. Typically the solvent for oxidation withhydrogen peroxide is glacial acetic acid. Precipitate formed during thesodium periodate oxidation is removed by filtration and the productconcentrated.

4.2.4. Protection of the Amine Group

The present invention further provides for protection of the amine groupafter synthesis of the benzhydrylamine. Protection of the amine groupcan be important to prevent reactivity of the amine when coupling thehandle to the solid support. Thus the invention provides amine-protectedhandles.

Preferred protecting groups for the amine are Boc and Fmoc. However, theother protecting groups, such as are described in Greene and Wuts,Protective Groups in Organic Synthesis, Wiley Interscience: New York,1991, pp. 309-362; and Fields and Noble 1990, Int. J. Pept. Protein Res.35: 161-214 are also contemplated. The synthesis of Boc and Fmocprotected amines is straightforward and well known in the art. N.sup.α-protected amino acids for peptide synthesis are usually protected withBoc or with Fmoc, depending on the synthetic, strategy.

In a specific embodiment, infra, the amine is protected with an Fmocprotecting group. The pH of the aqueous solution containingbenzhydrylamine is brought to about 9, any inorganic salts that haveprecipitated are removed by filtration, and protected Fmoc (e.g.,Fmoc-H-N-hydroxy-succinimidyl ester) added. The Fmoc-benzhydrylamine isworked up using standard procedures to yield a product of desiredpurity.

In an alternative embodiment, the benzhydrylamine is protected with theBoc protecting group. Generally, this can be accomplished according toroutine methods.

4.2.5 Alternate Route for Preparing the Handle

A benzophenone is prepared as described in Section 4.2.1., supra. Thebenzophenone can be reduced, e.g., by treatment with sodium borohydridein ethanol or other similar solvent, to yield benzhydryl alcohol.Treatment of the benzhydryl alcohol with a primary amide under acidicconditions results in substitution of the amide for the hydroxy group.This result is analogous to an acylation of a benzhydrylamine.

Preferably the amide group that is chosen is an N.sup.α -protected aminoacid, as described in Section 4.1., supra.

4.3. USE OF THE HANDLE FOR PEPTIDE SYNTHESIS

The handles of this invention are well suited for attaching a peptidechain to a solid phase support for peptide synthesis. The handles can beattached to any amino resins via a suitable functional group, e.g.,carboxylic acid, on the alkyl group. Attachment of a carboxylic acidfunctional group to an amine on the resin can proceed according to anyof the techniques commonly used for peptide synthesis, e.g., preparationof an OPfp, HOBt or other activated ester, condensation in the presenceof a carbodiimide, etc. These methods are also discussed for synthesisof peptides in Section 4.3.2. Suitable solid supports for use in theinvention are discussed, supra.

Solid phase peptide synthesis techniques are well known in the art.Simply put, an N.sup.α -protected amino acid is activated at theα-carbonyl and coupled with the deprotected N.sup.α of the nascentpeptide-handle-solid phase support. The coupling reactions may beaccomplished by techniques familiar to those in the art (see, e.g.,Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford, Ill.; Fields and Noble, 1990, "Solid phasepeptide synthesis utilizing 9-fluorenylmethyloxycarbonyl amino acids,"Int. J. Pept. Protein Res. 35:161-214; Geysen et al., 1987, J. Immunol.Methods 102:259-274). The chemistry of coupling, deprotection, andfinally cleavage of the peptide from the solid phase support depends onchoice of .sup.α N-protecting group, which is generallytert-butoxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc).

In a preferred embodiment of peptide synthesis using handles of theinvention the completeness of coupling should be assessed. Methods ofassessing the completeness of coupling are well known in the art. If thecoupling was not complete, the reaction should be forced to completionby a second coupling, e.g., (a) by using a higher concentration of theactivated amino acid or a different activation mechanism; (b) with theaddition of different or additional solvents; or (c) with the additionof chaotropic salts (see Klis and Stewart, 1990, in Peptides: Chemistry,Structure and Biology, Rivier and Marshall (eds.), EDSCOM Publishers,pp. 904-906).

4.3.1. Reduction and Cleavage

After peptide synthesis, the peptide must be cleaved from the solidphase support. If the handle of the invention is in its reduced form,cleavage is accomplished by treatment with acid, for example TFA. WhereFmoc amino acid strategy has been employed, the cleavage will alsoresult in deprotection of the side chains.

Alternatively, when the handle has been used in the oxidized form,either sulfoxide or sulfone, the handle can be reduced to convert it toacid labile handle. Reduction agents known in the art can be used. Forexample, about 1Mtrimethylsilylchloride/triphenylphosphine/tetrahydrofuran or morepreferably 1M trimethylsilylchloride/triphenylphosphine/dichloromethanecan be used to reduce the sulfoxide to the sulfide. Treatment withsamarium-iodide (SmI₂) will reduce the sulfone and sulfoxide to thesulfide. In yet a further embodiment, the reduction, deprotection andcleavage can be accomplished simultaneously by treatment with silylbromide. In another embodiment, reduction, deprotection (of Fmoc aminoacids) and cleavage can be accomplished simultaneously by treatment with1M trimethylsilylbromide/thioanisole/trifluoracetic acid.

A particular advantage of the instant invention is that the amino acidside chains can be deprotected prior to cleavage. In a specificembodiment, infra, the deprotected side chains react to form a cyclicpeptide prior to cleavage (see Sections 7.3 and 8., infra). By cyclizingprior to cleavage, side reactions and intermolecular bonding is avoided.

5. EXAMPLE Preparation of a Solid Support With a N-Fmoc2-(carboxylatobutyl-4oxy)-4-(methylsulfinyl) phenyl!- 4'(methylsulfinyl)phenyl! methylamine

5.1. 4.4'-bis(methylthio)-2-hydroxybenzophenone

A solution of 2-hydroxy-4-methylthiobenzoic acid (45.00 g, 227 mmol) inthionyl chloride (50 ml, 685 mmol) was heated at 40° C. for 15 min withstirring. After addition of 1,2-dichloroethane (EDC) (90 ml) thesolution was stirred at 50° C. for 30 min under vacuum (15 mm).

The solid residue was dissolved in EDC (250 ml) at 40° C. and added tothe solution of thioanisole (26.7 ml, 227 mmol) in EDC (100 ml). Theresulting mixture was cooled to 0° C. and finely ground AlCl₃ (33.0 g,247 mmol) was added portionwise within 75 min at 0°-5° C. The mixturewas allowed to warm to 20° C. and the second equivalent of AlCl₃ (33.7g, 253 mmol) was added within 30 min (caution: the temperature rises to30° C. and the mixture must be cooled). When addition of AlCl₃ wasfinished, the reaction mixture was stirred at 45°-50 ° C. for 1.5 hr,then cooled down to 20° C. and poured into mixture of ice (300 g) andconc. HCl (180 ml). The organic phase was evaporated and the aqueouslayer was heated at reflux for 15 min, cooled down to 20° C. andextracted with EDC (4×150 ml). The collected organic phases were washedwith water (3×50 ml), 10% NaHCO₃ (2×150 ml), water (1×150 ml), 1M HCl(1×150 ml) and water (2×150 ml) and then were dried over MgSO₄. Removalof the solvent in vacuo gave yellow crystals which were recrystallizedfrom EtOH (2100 ml) to give a yellow product (41.00 g, 62%), m.p.104°-106° C. Anal. Calcd for C₁₅ H₁₄ O₂ S₂ (290.41); C 62.04%, H 4.86%,S 22.08%. Found: C 61.86%, H 4.78%, S 22.06%. ¹ H NMR (CDCl₃): 2.51 s(3H, SCH₃); 2.54 s (3H, SCH₃); 6.60-6.85m (2H, aromatic); 7.26-7.66m(5H, aromatic); 12.35 s (1H, OH). MS-EI: 290(M⁺,100); 275(20); 243(30);228(10); 167(40); 151(30); 124(50); 105(10); 77(10); 57(10); 45(10). IR(CCl₄): ν_(CO) 1620 cm⁻¹ ; ν_(OH) 3000 cm⁻¹.

5.2. 5- 3-methylthio-1-oxy-5-(4-methylthiobenzoyl)phenyl!-pentanoic AcidMethylester

A suspension of alumina (90 g, Fluka-Type 504 C, acid type) andpotassium fluoride (dihydrate) (60 g) in water (600 ml) was evaporatedto dryness in vacuo. The residue was dried at 135°-140° C. for 24h toafford 120 g of KF/Al₂ O₃.

4,4'-Bis(methylthio)-2-hydroxybenzophenone (50.54 g, 174 mmol) wasdissolved in acetonitrile (800 ml) at 60° C. The solution was rapidlycooled down to 25° C. and KF/Al₂ O₃ (127 g) was added all at once. Tothe stirred suspension methyl bromovalerate (32.4 ml, 226 mmol) wasadded portionwise within 90 hr and stirring was continued for 120 hr atwhich time RP HPLC analysis indicated the reaction was complete (VydacC-18 column, isocratic eluent 75% MeOH, 0.1% TFA, starting compoundR_(t) =16 min, product R_(t) =7 min). The suspension was filtered,filtrate cake washed with acetone (2×50 ml) and the filtrateconcentrated in vacuo to give yellow product which was used in the nextstep without further purification. Analytical sample: m.p. 61°-63° C.(EtOH); R_(f) 0.45 (petroleum ether:EtOAc--60:40). Anal. 61°-63° C.(EtOH); R_(f) 0.45 (petroleum ether:EtoAc--60:40). Anal. Calcd for C₂₁H₂₄ O₄ S₂ (404.55): C 62.35%, H 5.98%, S 15.85%. Found; C 62.20%, H6.02%, S 15.62%. ¹ H NMR (CDCl₃): 1.25-1.55m (4H, 2×CH₂); 2.15 t (2H,CH₂ COO, J³ =7.5 Hz); 2.52 s (3H, SCH₃); 2.53 s (3H, SCH₃); 3.64 s (3H,COOCH₃); 3.90 t (2H, O₄ CH₂, J³ =5 Hz); 6.79 d (1H, J³ =1.5 Hz,aromatic); 6.88 dd (1H, J³ =8 Hz), J⁴ =1.5 Hz, aromatic); 7.18-7.25 m(2H, aromatic); 7.36 d (1H, J³ =8 Hz, J⁴ =1.5 Hz, aromatic); 7.18-7.25m(2H, aromatic); 7.36 d (1H, J³ =8 Hz, aromatic); 7.64-7.71m (2Haromatic) EI-MS: 404 (M⁺,30); 289(20); 227(40); 167(35); 151(35);124(20); 115(100); 83(20); 55(40).

5.3. 5- 3-methylthio-1-oxy-5-(4-methylthiobenzoyl)phenyl!-pentanoic Acid

The above prepared crude 5-3-methylthio-1-oxy-5(4-methylthiobenzoyl)phenyl!pentanoic acidmethylester was dissolved in dioxane (650 ml) and to this solution, 4MNaOH (131 ml, 522 mmol) and methanol (200 ml) were added. The resultingclear solution was stirred overnight at room temperature and then theorganic solvents were evaporated in vacuo. The aqueous solution wasextracted with ethyl acetate (EtOAc) (2×100 ml) and the aqueous layerwas kept in vacuo for 30 min to remove traces of EtOAc. The product wasprecipitated by slow addition of 20% H₂ SO₄ (to pH 2), washed with waterand dried over P₂ O₅ in vacuo to give an off-white powder (50.18 g, 87%,based on starting 4,4'-bis(methylthio)-2-hydroxybenzophenone).Analytical sample: m.p. 113°-114° C. (EtOH); R_(f) 0.22 (petroleumether:EtOAc--60:40). Anal. Calcd for C₂₀ H₂₂ O₄ S₂ (390.52): C 61.55%, H5.68%, S 16.42%. Found: C 61.15%, H 5.79%, S 16.39%. EI-MS: 390 (M⁺,50); 290(90); 243(50); 227(40); 167(100); 151(70); 124(95), 55(60).

5.4. 2-(carboxylatobutyl-4-oxy)-4-methylthio-4'-methylthiobenzophenoneOxime

A suspension of 5-3-methylthio-1-oxy-5-(4methylthio-benzoyl)phenyl!pentanoic acid (18.00g, 46.1 mmol), anhydrous sodium acetate (9.45 g, 115 mmol) andhydroxylamine hydrochloride (9.60 g, 138 mmol) in 96% ethanol (240 ml)was heated at reflux for 7 hr. After standing overnight at roomtemperature, the mixture was filtered and concentrated in vacuo to givea white solid, which was used without further purification in the nextstep. Analytical sample: m.p. 166°-170° C. (EtOH, isomeric mixture);R_(f) 0.41 (CHCl₃ :eOH--10:1). EI-MS: 405(M⁺, 2), 387 (25); 300(45);288(100); 242(30); 227(20); 168(30); 151(40); 124(10); 55(35).

5.5.2-(carboxylatobutyl-4-oxy)-4-(methythio)phenyl-4'-methylthiophenyl!methylamine4-toluenesulfonate

Crude 2-(carboxylatobutyl-4-oxy)-4-methylthio-4'-methylthio-benzophenoneoxime (minimum 46.1 mmol) prepared above was placed in a 500 mlChampagne bottle together with powdered zinc (18.37 g, 461 mmol),ethanol (80 ml) and 25% ammonia (240 ml). The suspension wasmagnetically stirred at 50° C. for 48 hr. After filtration, the filtratewas concentrated in vacuo to give a white solid. After addition of water(300 ml) pH was adjusted to 7 with a saturated solution of toluenesulfonic acid (TsOH) in EtOH and the suspension was filtered. Theoff-white product was washed with water and then suspended in ethanol(300 ml) at about 60° C. To this suspension, the saturated solution ofTsOH in EtOH was added dropwise until the mixture turned to a clearorange solution. Standing in a refrigerator for 48 hr afforded pinkcrystals, which were suspended by filtration to give the product 1 (18.2g, 70%), m.p. 178°-180° C. (dec); R_(f) 0.58 (n-BuOH:AcOH:H₂ O--80:20:20). FAB-MS: 392(m+1); 375; 275; 227; 137. Anal calcd for C₂₇H₃₃ NO₆ S₃ (563.76); C 57.52%, H 5.9%, N 2.48%, S 17.06%. Found: C57.87%, H 5.67%, N 2.40%, S 17.11%.

5.6. N-9-Fmoc-{2-carboxylatobutyl-4-oxy)-4-(methylsulfinyl)phenyl-4'-(methylsulfinyl)-phenyl}methylamine

The2-(carboxylatobutyl-4-oxy)-4-(methylthio)phenyl-4'-methylthiophenyl!methylamine4-toluenesulfonate (20.00 g, 35.5 mmol) was suspended in the mixture ofmethanol (320 ml) and water (160 ml). The suspension was heated untilthe solution became clear, then chilled to 20° C. and a solution ofsodium periodate (15.18 g, 71 mmol) in water (140 ml) was added dropwiseover a period of 1 hr. The stirring was continued further for 2 hr. Thesodium iodate which separated was removed by filtration and the methanolwas evaporated in vacuo. The pH of remaining aqueous solution wasadjusted to 9 by addition of 4M NaOH and the solution was mixed withacetonitrile (200 ml). The mixture was filtered to remove precipitatedinorganic salts and a solution offluorenylmethyloxycarbonyl-N-hydroxysuccinimidate (Fmoc-succinimidate)(12.99 g, 38.5 mmol) in acetonitrile (80 ml) was added rapidly. Theresultant homogenous solution was stirred at 25° C. while adjusting thepH to 8.5-9 with 1M NaOH. After 2 hr no further pH change occurred andthe acetonitrile was removed in vacuo. The remaining aqueous solutionwas diluted with water (100 ml) and extracted with ether (4×50 ml,discarded). The aqueous part was mixed with chloroform (150 ml) and 1MH₂ SO₄ was added with occasional shaking until the aqueous phase reachedpH 2. The aqueous phase was then extracted with chloroform (4×50 ml).The combined organic phases were washed with water (100 ml), dried(MgSO₄), and concentrated to give an oil which after dissolving inmethanol (150 ml) and standing overnight in refrigerator afforded theproduct as a white solid. Concentration of mother liquor and addition ofethyl acetate (50 ml) gave the second portion of the product. Yield14.75 g (65%), m.p.--135°-137° C. R_(f) (CHCl₃ :MeOH--9:1)=0.11. ¹ H NMR(CDCl₃): 1.3-1.6m (4H, CH₂), 2.17 t (2H, CH₂ COO, J=8 Hz), 2.75 s (3H,SOCH₃), 2.76 s (3H, SOCH₃), 3.8-4.05m (2H, OCH₂), 4.22 t (1H, Fmoc-CH,J=8 Hz), 4.4-4.6m (2H, CH₂ -Fmoc), 5.95 d (1H, Ar₂ CH, J=9 Hz), 6.25 d(1H, NH, J=9 Hz), 7.10-7.80m (15H, aromatic). Anal Calcd for C₃₅ H₃₅ NO₇S₂ (645.8): C 68.10%, H 5.46%, N 2.17%, S 9.93%. Found: C 68.55%, H5.50%, N 2.11%, S 9.89%.

6. EXAMPLE Attachment of Handle onto Support

The solution on N-Fmoc-{2-(carboxylatobutyl-4oxy)-4-(methylsulfinyl)phenyl!-4'-(methylsulfinyl)-phenyl}methylamine(245 mg, 0.38 mmol), N-hydroxybenzotriazole (52 mg, 0.38 mmol),4-dimethylaminopyridine (5 mg, 0.038 mmol), andN,N'-diisopropylcarbodiimide (50 μl, 0.38 mmol) in dimethylformamide (10ml) was added to p-methylbenzhydrylamine (pMBHA) resin (200 mg, 0.076mmol) (Peptides International, Louisville, Ky.) and the suspension wasshaked overnight. The resin was washed with dimethylformamide (5×5 ml)and allowed to react with the solution of acetic acid (46 μl, 0.76 mmol)and N,N'-diisopropylcarbodiimide (119 μl, 0.76 mmol) indimethylformamide (10 ml) for 5 hr to acetylate the remaining free aminogroups. After washing with dimethylformamide (5×5 ml), dichloromethane(5×5 ml), and methanol (5×5 ml) the resin was dried in vacuo. Thesubstitution estimated spectrophotometrically, was 0.34 mmol/g.

7. EXAMPLE Preparation of Peptides Using the Handle

7.1. H-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH₂

The above prepared resin (132 mg) was used. The general syntheticprotocol was as follows: The Fmoc protecting group on the handle wasremoved with piperidine/dimethylformamide (1:1, v/v, 1×3 min, 1×10 min)followed by washing with dimethylformamide. Boc protecting groups wereremoved with mixture of trifluoroacetic acid/dichloromethane/anisole(50:50:1, 1×3 min, 1×25 min) followed by washing with dichloromethane.N-Hydroxybenzotriazole active esters were used throughout all synthesis(10 min pre-activation). The Boc protecting group was used in the casesof Gly, Val, and Ile amino acids. In the other cases, the Fmoc group wasused. The coupling was monitored with bromophenol blue (Krchnak et al.,1988, Collect. Czech. Chem. Commun. 53:2542). Final cleavage fromsupport was accomplished with the mixture of1Mtrimethylsilylbromide/thioanisole/trifluoroacetic acid (2 hr, 0° C.)followed by dilution with water and extraction with diethyl ether. Thecrude peptide was obtained in 95% yield (43 mg) and was purified onSephadex G 25 with 1M acetic acid as eluent. After preparative reversephase HPLC the pure peptide was obtained in 62% yield (26.6 mg). FAB-MS:1056 (M+1), 942, 871, 814, 715, 658, 545, 474, 391, 245, 217, 120. Aminoacid analysis: Thr (1.00), Gln (1.02), Pro (1.97), Gly (2.00), Ala(1.95), Val (0.97), Ile (0.94), Phe (1.01).

7.2. Boc-Tyr-Ile-Gln-Asn-Cys(Clz-Abu-O^(T) Bu)-Pro-Leu-Gly-Handle-pMBHAResin

pMBHA resin prepared analogously as in Example 6 (400 mg, 0.052 mmol)was used for the synthesis of C¹ -OXT. After each coupling, theremaining free amino groups were acetylated with the mixture of aceticacid/N-hydroxybenzotriazole/N,N'-diisopropylcarbodiimide/N-methylimidazole.The general synthetic protocol used was the same as described in 8.1,supra. All amino acids used were Fmoc-derivatives. Amino acid analysis:Asp(0.98), Glu(1.00), Pro(0.94), Gly(1.13), Cyth(0.54), Ile(0.90),Leu(1.04), Tyr(0.75).

7.3 ##STR3##

Above prepared peptidyl-resin (Section 7.2, supra) (100 mg, 0.012 mmol)was washed with dichloromethane (3×5 ml) and tert-butyl protectinggroups were removed with mixture of trifluoroaceticacid/dichloromethane/anisole (50:50:1, 1×3 min, 1×25 min), followed bywashing with dichloromethane (5×5 ml), neutralized with 7%N,N'-diisopropylcarbodiimide/dichloromethane, washed withdichloromethane (3×5 ml) and finally washed with N,N-dimethylformamide(3×5 ml). The cyclization was carried out on the support withN-hydroxybenzotriazole/N,N'-diisopropylcarbodiimide/N,N-dimethylformamidemixture for 24 hr to give a cyclized, fully protected peptide. The finalcleavage was accomplished with 1Mtrimethylsilylbromide/thioanisole/trifluoroacetic acid mixturecontaining 5% of m-cresole (2 hr, 0° C.). The peptide was precipitatedwith diethyl ether, filtered, washed with diethyl ether and purified onSephadex G 25 (1M acetic acid) to give 10.6 mg (89%) of crude product,which was purified on preparative RP-HPLC (Vydac C18) to give 4.5 mg(38%) of a pure C¹ -OXT. Amino acid analysis: Asp(0.99), Glu(1.00),Pro(0.95), Gly(0.99), Cyth(1.02), Ile(0.96), Leu(1.10), Tyr(0.93),MS(FAB)=990 (M+H).

8. EXAMPLE Two-Step Deprotection of C¹ -OXT Precursor

Above prepared peptidyl-resin (Section 7.2, supra) (100 mg, 0.012 mmol)was treated with the mixture of 1Mtrimethylsilylchloride/triphenylphosphine/tetrahydrofuran (2 ml) at 25°C. for 2 hr. Then the resin was washed with dichloromethane (5×5 ml) andthe peptide was cleaved from the resin with the mixture oftrifluoroacetic acid/m-cresole (95:5) (2.5 ml) at 25° C. for 30 min.After evaporation to dryness, the peptide was precipitated with diethylether to give a powder which was purified on Sephadex G 25 (1M aceticacid) followed by preparative RP-HPLC (Vydac C18) to give 6.5 mg (43%)of a pure H₂ N-Tyr-Ile-Gln-Asn-Cys(ClZ-Abu-OH)-Pro-Leu-Gly-NH₂. Aminoacid analysis: Asp(1.04), Glu(1.04), Pro(0.95), Gly(1.03), Cyth(0.94),Ile(0.88), Leu(1.11), Tyr(0.85), MS(FAB)=1176 (M+H).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A handle for use in peptide synthesis of thegeneral formula (I) ##STR4## wherein the phenyl group bearing (X¹)_(a),Z and optionally (Y)_(c) is referred to as a first phenyl group, and thephenyl group bearing (X²)_(b) and optionally (Y)_(d) is referred to as asecond phenyl group;X¹ is selected from the group consisting of --SR¹,--S(O)R¹, and --S(O)₂ R¹, each X¹ being in an ortho or para position,with respect to the carbon atom of the handle bearing NHD, on the firstphenyl group; R¹ is a C₁ -C₁₀ hydrocarbon group; Z is --OR³, being in anortho or para position, with respect to the carbon atom of the handlebearing NHD, on the first phenyl group; R³ is a C₁ -C₁₀ hydrocarbongroup substituted with a carboxyl group for coupling to a solid phasesupport; X² is selected from the group consisting of --SR², --S(O)R²,and --S(O)₂ R², each X² being in an ortho or para position, with respectto the carbon atom of the handle bearing NHD, on the second phenylgroup; R² is a C₁ -C₁₀ hydrocarbon group; Y is --OR⁴, being in an orthoor para position, with respect to the carbon atom of the handle bearingNHD, not occupied by (X¹)_(a) or Z on the first phenyl group and in anortho or para position, with respect to the carbon atom of the handlebearing NHD, not occupied by (X²)_(b) on the second phenyl group; R⁴ isa C₁ -C₁₀ hydrocarbon group; a=1-2; b=1-3; c=0-1 d=0-1; and D isselected from the group consisting of --H, a protecting group and anN.sup.α -protected acyl: wherein(i) said --S(O)R² and --S(O)₂ R² groupsare convertible to --SR² groups upon treatment with a mixture oftriphenylphosphine and trimethylsilyl halide; and (ii) said mixture oftriphenylphosphine and trimethylsilyl halide does not cleave said NHDfrom said handle.
 2. The handle of claim 1 wherein X² is --S(O)₂ R². 3.The handle of claim 1 whereina=1; (X¹)_(a) is in the para position, withrespect to the carbon atom of the handle bearing NHD, on the firstphenyl group; b=1; and (X²)_(b) is in the para position, with respect tothe carbon atom of the handle bearing NHD, on the second phenyl group.4. The handle of claim 1 wherein X¹ is --SR¹ and X² is --SR².
 5. Thehandle of claim 1 wherein X¹ is selected from the group consisting of--S(O)R¹ and --S(O)₂ R¹, and X² is selected from the group consisting of--S(O)R² and --S(O)₂ R².
 6. The handle of claim 1 wherein D is aprotecting group.
 7. The handle of claim 6 wherein said protecting groupis selected from the group consisting of tert-butoxycarbonyl, andN-9-fluorenylmethyloxy carbonyl.
 8. The handle of claim 1 in whereinc=1,d=0; and (Y)_(c) is in an ortho position, with respect to the carbonatom of the handle bearing NHD, on the first phenyl group.
 9. The handleof claim 1 wherein c=0, d=1 and (Y)_(d) is in an ortho position, withrespect to the carbon atom of the handle bearing NHD, on the secondphenyl group.
 10. A handle for use in peptide synthesis of the generalformula: ##STR5## wherein Z is --OR⁴,R⁴ is a C₁ -C₁₀ hydrocarbon groupcomprising a carboxyl group for coupling to a solid phase support; R¹ isa C₁ -C₁₀ hydrocarbon group and in which each G is independentlyselected from the group consisting of --S--, --SO--, and --SO₂ --; D isselected from the group consisting of H, a protecting group and anN.sup.α -protected acylamine; said --SO-- and --S(O)₂ -- groups areconvertible to --S-- groups upon treatment with a mixture oftriphenylphosphine and trimethylsilyl halide; and said mixture oftriphenylphosphine and trimethylsilyl halide does not cleave said NHDfrom said handle.